Project Summary/Abstract: The mechanistic target of rapamycin complex 1 (mTORC1) serves a critically central role in cellular growth and metabolism by integrating many diverse inputs that signal nutrient availability and hormonal cues to the cell. In response to these stimuli, mTORC1 governs cellular metabolism and growth by regulating anabolic and catabolic programs like protein synthesis and autophagy, respectively. Correspondingly, dysfunctional mTORC1 signaling has been implicated in many human diseases like cancer. Activation of mTORC1 signaling by growth factors such as insulin has been heavily studied and characterized. In recent years, sensing of intracellular amino acids has also been discovered to be essential for mTORC1 recruitment to the lysosomal membrane, which is required for its activation. In cell lines, much of the mechanism of leucine sensing by the mTOR pathway has been elucidated. Leucine sensing is mediated by Sestrin1 and Sestrin2, whose inhibitory interactions with mTOR pathway components are relieved by leucine binding. However, amino acid sensing has yet to be mechanistically explored as a component of mTORC1 activation in tissues, including in the liver. An understanding of leucine sensing in vivo offers a selective approach of pharmacologically targeting mTORC1 activity that bypasses growth factor signaling. This may enable development of improved calorie restriction mimetics and would provide valuable treatment options for pathologies resulting from abnormal leucine levels, such as maple syrup urine disease (MSUD). I will compare mTORC1 responsiveness to leucine in the livers of wild-type mice, Sestrin knockout mice, and mice expressing a Sestrin2 mutant (W444L) with reduced leucine affinity. Using imaging and biochemical assays, I will identify the mechanism of leucine sensing in the liver and the role of Sestrins in this process. As preliminary data indicate, mTORC1 is stimulated by leucine in mouse liver, highlighting an unknown cellular sensing mechanism in the liver. The goal of the proposed project is to interrogate the role of leucine sensing in hepatic mTORC1 activation and liver function in vivo and determine the involvement of Sestrins in this process. I propose the following aims: 1. Determine how leucine availability regulates mTORC1 activity in mouse liver. 2. Establish a role of leucine sensing by Sestrins in mTORC1 activity in vivo. 3. Identify the contribution Sestrin-mediated leucine sensing in liver function. The proposed study will reveal a currently unknown mechanism of leucine sensing by the mTOR pathway in vivo and identify its role in liver function. An understanding of in vivo leucine sensing will be valuable for potential development of improved calorie restriction mimetics and for informing therapeutic intervention for patients suffering from disorders of leucine homeostasis.